J.R. Bamburg will use the epi-fluorescence microscope with stage incubator for the microinjection of fluorescently labelled proteins into living cells. The image intensification and analysis system will be utilized to follow the distribution of fluorescently labelled cytoskeletal proteins into their cellular components. The dynamics of actin and actin depolymerizing factor in the neuronal growth cone will be observed during the transition from active neurite outgrowth to the quiescent state. Subcellular distribution of calcium ion will be measured to correlate changes in calcium with alterations seen in the organization and assembly of actin. The linear photodiode array camera and intensity stabilized light table will be used to identify, by two-dimensional (2-D) gel analysis, proteins whose interaction with the growth cone cytoskeleton is modulated by agents which alter growth. J.S. Bedford will use the epi-fluorescence microscope to measure the proliferative status of irradiated cells by a new immunofluorescent assay for DNA polymerase alpha. The image analysis system coupled to the fluorescence microscope will be used to analyze interphase chromosomal damage and the early stages of chromosomal aberration formation following radiation exposure. The 2-D gel analysis system is required for examining the differences which exist in the proteins synthesized in thermotolerantless mutants of CHO cells which have been isolated. R.J. Gillies will use the instrumentation to discern the spatial and temporal subcellular distribution of both hydronium and calcium ions in mouse fibroblasts. These ions will be monitored during the cells proliferative response to serum or growth factors. The effects of these agents on the assembly of actin in these cells will also be monitored by cytochemistry of fluorescent analogs. The 2-D gel analysis system will be used to identify proteins that have been synthesized in response to mitogenic signals. M.R. Paule will use the microscope and microinjection system for transformation of Acanthamoeba. The image analysis system will be used for identification of modified RNA polymerase subunits on 2-D gels, for quantitative transcription assays and footprinting, and for DNA sequence analysis using the molecular biology workstation.